DNA cloning technology provides a readily amplifiable source of genes encoding any protein of interest. When the recombinant protein itself is needed, genes are cloned in expression vectors that are introduced into appropriate cell types where protein synthesis can take place. To produce large amounts of these proteins, two general types of transient gene expression vectors have been used: plasmid DNA vectors which are introduced directly into cells and viral vectors that express foreign genes as part of their genetic material. The latter type of vector is generally more efficient in higher eukaryotic cells because all cells can be infected simultaneously and many viruses can express proteins at very high levels. Plasmid vector preparation is less labor intensive but DNA transfection can be inherently less efficient and amounts of protein synthesized are generally lower.
High-level recombinant protein expression is crucial for the biopharmaceutical industry as well as for basic research. Large amounts of specific proteins are very often required for general biochemical characterization, structural studies, drug discovery development, gene therapy, subunit vaccine production, and reagent use. Different uses dictate which particular protein expression system provides the best combination of properties. For example, high-level transient protein expression in mammalian cells most often makes use of viral vectors (e.g., adenovirus, baculovirus, poxvirus, alphavirus). In most applications, the gene of interest is cloned into the virus genome or a derivative replicon which is labor intensive and time consuming. Plasmid vectors are also used for transient protein expression but efficiency is generally much lower. Another method employs recombinant viruses that express the T7 RNA polymerase to drive expression of desired proteins from plasmids under control of a T7 promoter. This latter approach is very efficient using a vaccinia-T7 recombinant virus especially when incorporating an internal ribosome entry sequence (IRES) in the T7 transcript. However, high level protein production using the vaccinia-T7 system is limited to host cells that grow the virus efficiently. Moreover, the use of an infectious virus related to the smallpox vaccine strain raises biosafety concerns.
In the last few years, advances in nucleic acid chemistry and gene transfer have inspired new approaches to engineer specific interference with gene expression. Antisense technology has been the most commonly described approach in protocols to achieve gene-specific interference. For antisense strategies, stoichiometric amounts of single-stranded nucleic acid complementary to the messenger RNA for the gene of interest are introduced into the cell. Some difficulties with antisense-based approaches relate to delivery, stability, and dose requirements. In general, cells do not have an uptake mechanism for single-stranded nucleic acids, hence uptake of unmodified single-stranded material is extremely inefficient. While waiting for uptake into cells, the single-stranded material is subject to degradation. Because antisense interference requires that the interfering material accumulate at a relatively high concentration (at or above the concentration of endogenous mRNA), the amount required to be delivered is a major constraint on efficacy. As a consequence, much of the effort in developing antisense technology has been focused on the production of modified nucleic acids that are both stable to nuclease digestion and able to diffuse readily into cells.
Double-stranded RNA (dsRNA) can induce sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). However, in mammalian cells, dsRNA that is 30 base pairs or longer can induce sequence-nonspecific responses that trigger a shut-down of protein synthesis. Recent work suggests that RNA fragments are the sequence-specific mediators of RNAi (Elbashir et al., Nature 2001 411:494; Elbashir et al., Genes and Development 2001, 15:188). Interference of gene expression by these small interfering RNA (siRNA) is now recognized as a naturally occurring strategy for silencing genes in C. elegans, Drosophila, plants, and in mouse embryonic stem cells, oocytes and early embryos (Cogoni et al., Antonie Van Leeuwenhoek 1994, 65:205; Baulcombe, Plant Mol. Biol., 1996, 32:79; Kennerdell and Carthew, Cell 1998, 95:1017; Timmons and Fire, Nature 1998, 395:854; Waterhouse et al., Proc. Natl. Acad. Sci. U.S.A. 1998, 95:13959; Wianny and Zernicka-Goetz, Nat. Cell Biol. 2000, 2:70; Yang et al., Mol. Cell Biol. 2001, 21:7807; Svoboda et al., Development 2000, 127:4147 (2000). In mammalian cell culture, a siRNA-mediated reduction in gene expression has been accomplished by transfecting cells with synthetic RNA oligonucleotides (Caplan et al., Proc. Natl. Acad. Sci. U.S.A. 2001, 98:9742; Elbashir et al., 2001, supra). However, as Bass (Nature 2001, 411:428) notes, various issues regarding the use of siRNA in mammalian cells have yet to be addressed, including effective delivery of siRNA to mammalian cells in vivo. Furthermore, if siRNA is to be utilized in in vivo therapy, it will be important in many cases to develop methods to express siRNA in tissues in vivo to achieve extended intracellular transcription of the siRNA.
Vesicular stomatitis virus (VSV), of the genus, Vesiculovirus, is the prototypic member of the family Rhabdoviridae, and is an enveloped virus with a negative stranded RNA genome that causes a self-limiting disease in live-stock and is essentially non-pathogenic in humans. Balachandran and Barber (2000, IUBMB Life 50: 135-8). Rhabdoviruses have single, negative-strand RNA genomes of 11,000 to 12,000 nucleotides (Rose and Schubert, 1987, Rhabdovirus genomes and their products, in The Viruses: The Rhabdoviruses, Plenum Publishing Corp., NY, pp. 129-166). The virus particles contain a helical, nucleocapsid core composed of the genomic RNA and protein. Generally, three proteins, termed N (nucleocapsid, which encases the genome tightly), P (formerly termed NS, originally indicating nonstructural), and L (large) are found to be associated with the nucleocapsid. An additional matrix (M) protein lies within the membrane envelope, perhaps interacting both with the membrane and the nucleocapsid core. A single glycoprotein (G) species spans the membrane and forms the spikes on the surface of the virus particle. Glycoprotein G is responsible for binding to cells and membrane fusion. The VSV genome is the negative sense (i.e., complementary to the RNA sequence (positive sense) that functions as mRNA to directly produce encoded protein), and rhabdoviruses must encode and package an RNA-dependent RNA polymerase in the virion (Baltimore et al., 1970, Proc. Natl. Acad. Sci. USA 66: 572-576), composed of the P and L proteins. This enzyme transcribes genomic RNA to make subgenomic mRNAs encoding the 5-6 viral proteins and also replicates full-length positive and negative sense RNAs. The genes are transcribed sequentially, starting at the 3′ end of the genomes.
VSV replicates rapidly (<12 hours) and very efficiently in the cytoplasm of almost all vertebrate cells and produces very high levels of infectious virus (titers approaching 20,000 infectious units/cell in some cases) and can also infect insect cells. The sequences of the VSV mRNAs and genome are described in Gallione et al. 1981, R Virol. 39: 529-535; Rose and Gallione, 1981, J. Virol. 39: 519-528; Rose and Schubert, 1987; Rhabdovirus genomes and their products, p. 129-166, in R. R. Wagner (ed.); The Rhabdoviruses, Plenum Publishing Corp., NY; Schubert et al., 1985, Proc. Natl. Acad. Sci. USA 82: 7984-7988). WO96/34625 published Nov. 7, 1996, disclose methods for the production and recovery of replicable vesiculovirus. U.S. Pat. No. 6,168,943, issued Jan. 2, 2001, describe methods for making recombinant vesiculoviruses.
Growth in tissue culture and purification of virus is relatively simple and methods for engineering mutations or additional genes in the virus genome, while retaining very high infectivity, are well established. It is moreover possible to engineer the surface protein of the virus and target infection to specific cell types. Natural hosts include cattle, horses, and pigs where it causes a non-fatal but debilitating disease. Laboratory strains pose very little if any risk of pathogenicity in humans. VSV is currently being explored as a vector for vaccine production, gene replacement therapy, and anticancer therapy.
Expression of the T7 RNA polymerase enzyme by recombinant viruses has been reported (see e.g. Mohammed et al., Methods Mol Biol. 2004; 269:41-50; and Eckert et al., J Gen Virol. 1999 June; 80 (Pt 6):1463-9). Recombinant T7-expressing viruses are capable of driving transient expression of proteins from plasmids. The best characterized of these systems is the recombinant vaccinia-T7 virus which yields very high levels or protein. The limitations and uses of any of the virus-T7 expression systems are in large part governed by the properties of the virus. Thus, there is a need for a safe and efficient alternative virus-T7 polypeptide expression system.